The ubiquitously expressed Na-H exchanger, NHE1, regulates intracellular pH (pHi) homeostasis and has a permissive effect in promoting cell growth. During the previous two funding cycles, the applicant has identified two serine/threonine kinases, ROCK and NIK, that act downstream of GTPase pathways to directly phosphorylate and activate NHE1. The current objective is to develop a comprehensive understanding of the functional significance of this regulation by investigating the hypothesis that divergent signaling networks converge on NHE1 to regulate cell growth and differentiation. This hypothesis will be investigated through specific aims that study upstream (Aim 1), direct (Aim 2), and downstream (Aim 3) components. In Aim 1, the upstream signaling events mediated by ROCK and NIK will be investigated to determine how signals from integrin and heptahelical receptors converge to regulate a ROCK-dependent activation of NHE1, and to identify receptors acting in an NIK-mediated pathway regulating NHE1. In Aim 2, the direct regulation of NHE1 will be investigated by determining whether NHE1 is a phosphosubstrate for serine/threonine phosphatases that are regulated by ROCK- or NIK-mediated pathways, and whether C-terminal modifications, including phosphorylation and the binding of regulatory proteins, promote the assembly of NHE1-associated signaling complexes and dynamic changes in NHE1 localization. In Aim 3, the significance of NHE1 in cell growth will be investigated by two approaches. Because the signaling pathways mediated by ROCK and NIK to activate NHE1 in mammalian cells are evolutionarily conserved as regulators of critical developmental stages, the applicant will use a genetic analysis to determine the functional importance of a novel Drosophila dnhe1 gene the applicant recently identified. Additionally, the applicant will identify downstream targets regulated by NHE1 activity in mammalian cells by using DNA arrays, and determine the functional importance of these targets in mediating the permissive effects of NHE1 on cell growth. Using new approaches with reagents the applicant developed during the previous two funding cycles, the applicant will answer significant questions that are critical for a comprehensive understanding of how NHE1 is regulated, and how it, in turn, regulates normal and pathological cell functions.